1. Field of the Invention
The present invention relates to disease detection and treatment for diseases such as cardiovascular and immune diseases and, more specifically, to a method for classifying patients at risk for cardiovascular disease, other chronic inflammatory diseases, and cardiovascular and non-cardiovascular mortality based on a risk assessment for lymphocyte activation gene 3 (LAG3) protein deficiency, and for mediating the risk using recombinant lymphocyte activation gene-3 as a companion therapeutic.
2. Description of the Background
It is now well-established that atherosclerosis is a chronic inflammatory disease, with coronary artery vessels infiltrated by innate and adaptive immune cells and cholesterol plaque that ultimately leads to vessel occlusion and clinical disease [Atherosclerosis—a matter of unresolved inflammation. Viola J, Soehnlein O. s.l.: Semin Immunol, 2015, Vol. 27, pp. 184-193]. A number of cardiovascular disease (CVD) risk factors are used clinically to assess atherosclerotic risk in patients, including hypertension, diabetes mellitus, lipids, age, and sex [Executive Summary. Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. s.l.: NIH Publication No. 01-3670, 2001]. In this era of precision medicine, it is understood that genetic causes also exert major influences on CVD risk. There are now a number of genome wide association studies (GWAS) that have identified known and novel loci that are significantly associated with CVD, especially diseases such as atherosclerosis and myocardial infarction [Genetics of human cardiovascular disease. Kathiresan S. Srivastava D. s.l.: Cell, 2012, Vol. 148, pp. 1242-1257].
It has been shown that the rs10846744 single nucleotide polymorphism (SNP) within the SR-B1 gene, SCARB1 (located on chromosome 12:q24.31), is significantly associated with subclinical atherosclerosis and CVD. In issued U.S. Pat. No. 9,334,538, the present inventor disclosed a method of genotyping women in order to identify the presence of the rs10846744 mutation of the SCARB1 gene (located on chromosome 12:q24.31). This was significantly associated with subclinical atherosclerosis (SCA) and incident cardiovascular disease (CVD) in participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Specifically, carriers of the risk C allele had significantly increased odds for incident CVD, and in a multivariable regression model this relationship was not attenuated by inclusion of traditional CVD risk factors such as age, body mass index, hypertension, smoking, renal disease, statin use or lipid levels (whether total cholesterol, LDL-cholesterol [LDL-C], HDL-C, or triglycerides). These findings strongly suggested that other factors or pathways might be causal in the association of this genetic variant with incident CVD.
Interestingly, rs10846744 resides within the first intron of SCARB1 and bioinformatic analysis revealed that this SNP resides within a regulatory region. The data suggested that this SNP could transcriptionally regulate genes on the same chromosome (intra-chromosomal) or inter-chromosomally. The present inventor investigated this possibility and a number of transcriptionally regulated gene candidates emerged. One in particular, lymphocyte activation gene-3 (LAG-3) is also located on chromosome 12 and was investigated further. LAG3 is a significant regulator in T lymphocyte activation [Lymphocyte-activation gene 3/major histocompatibility complex class II interaction modulates the antigenic response of CD4+ T lymphocytes. Huard B, Tournier M, Hercend T, Triebel F, Faure F. s.l.: Eur J Immunol, 1994, Vol. 24, pp. 3216-3221]. LAG3 belongs to the Ig superfamily and is a ligand to MHC class II molecules of antigen-presenting cells [LAG-3: a regulator of T-cell and DC responses and its use in therapeutic vaccination. F., Triebel. s.l.: TRENDS Immunol, 2003, Vol. 24, pp. 619-622]. It is expressed in B cells, T cells, and NK lymphocytes, monocytes, and dendritic cells (DC) and its major function is thought to be a negative regulator of activated T cells by controlling effector T cell expansion and homeostasis [LAG-3 regulates plasmacytoid dendritic cell homeostasis. Workman C J, Wang Y, El Kasmi K C, Pardoll D M, Murray P J, Drake C G, Vignali D A. s.l.: J Immunol, 2009, Vol. 182, pp. 1885-1891; The CD4-related molecule, LAG-3 (CD223), regulates the expansion of activated T cells. Workman C J, Vignali D A. s.l.: Eur J Immunol, 2003, Vol. 33, pp. 970-979]. Cell surface LAG3 is subject to cleavage by ADAM10 and ADAM17 metalloproteases, which results in soluble LAG3 (sLAG3) [Metalloproteases regulate T-cell proliferation and effector function via LAG-3. Li N, Wang Y, Forbes K, Vignali K M, Heale B S, Saftiq P, Hartmann D, Black R A, Rossi J J, Blobel C P, Dempsey P J, Workman C J, Vignali D A. s.l.: EMBO, 2007, Vol. 26, pp. 494-504].
In vitro and ex vivo approaches were taken to examine the association of rs10846744 with LAG3 in biospecimens isolated from hyperalphalipoproteinemic (HALP) subjects. It was found that rs10846744 is significantly associated with alterations in the expression and function of LAG3, and markers of intracellular inflammasomes such as NLRP3.
LAG3 is located near the CD4 loci on chromosome 12 (chr 12:p13) while rs10846744 is located on chr12:q24.32. LAG3 has a similar function, if not a competitive one against CD4, by binding MHC class II on antigen-presenting cells. [Sierro S, Romero P, Speiser D E. The CD4-like molecule LAG-3, biology and therapeutic applications, Expert Opin Ther Targets 2011; 15:91-101.]
Golden et al., Arteriosclerosis, Thrombosis, and Vascular Biology, 34: A359 (2014) has shown that human homozygous carriers of the SCARB1 rs10846744 risk (CC) allele had significantly lower plasma LAG3 protein levels. In vitro studies revealed that risk (CC) lymphocytes secreted more pro-inflammatory cytokines (TNFα) and less anti-inflammatory cytokines (IL-10) as compared with reference (GG) lymphocytes. Consequently, these same carriers of the risk (CC) allele were shown to have increased carotid intimal media thickness (cIMT), a known surrogate for CVD event risk.
In vitro and in vivo murine studies have suggested that sLAG3 regulates MHC class II signaling pathways to limit T cell activation and homeostasis, while a few clinical studies have shown associations between sLAG3 and tuberculosis resistance [Lienhardt et al, Active tuberculosis in Africa is associated with reduced Th1 and increased Th2 activity in vivo, G. s.l.: Eur J Immunol, 2002, Vol. 32, pp. 1605-1613] and breast cancer prognosis [Triebel et al., A soluble lymphocyte activation gene-2 (sLAG-3) protein as a prognostic factor in human breast cancer expressing estrogen or progesterone receptors. M-F. s.l.: Cancer Letters, 2006, Vol. 235, pp. 147-153]. In murine cells, Kisielow et al reported that activated T cells induced LAG3 expression on B cells. [Kisielow M, Kisielow J, Capoferri-Sollami g, Karjalainen K. Expression of lymphocyte activation gene 3 (LAG-3) on B cells is induced by T cells. Eur J Immunol 2005; 35:2081-2088.] They determined that LAG3 induction on B cells was T cell dependent and not dependent on other stimuli such as unmethylated CpG motif 1826, bacterial LPS, or anti-Ig antibody in combination with anti-CD40 and IL-4. In contrast, LAG3 RNA and protein was detected in EBV-transformed B cells, with significantly higher expression in EBV-transformed cells expressing the reference SCARB1 rs10846744 G allele as compared with cells expressing the risk C allele. Although EBV transformation of B lymphocytes could activate the cells, there was a significant difference in the level of LAG3 expression based on rs10846744 genotype stratification. Importantly, others observed a lack of LAG3 expression in B cells, such as Ramos cells [Baixeras E, Huard B, Miossec C, Jitsukawa S, Martin M, Hercend T, Auffray C, Triebel F, Piatier-Tonneau D., Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens. J Exp Med 1992; 176:327-337.] However, the present inventor genotyped these cells and found that they were heterozygous for the rs10846744 variant. More recently, Morales et al showed that EBV positivity in Hodgkin lymphomas were significantly associated with increased gene expression of LAG3. [Morales O, Mrizak D, Francois V, Mustapha R, Miroux C, Depil S, Decouvelaere A V. Lionne-Huyghe P, Auriauht C, de Launoit Y, Pancre V, Delhem N. Epstein-Barr virus infection induces an increase of T regulatory type 1 cells in Hodgkin lymphoma patients. Br J Haematol 2014 Jul. 9. Epub ahead of print]
Studies with murine models have shown that atherosclerotic lesion size and inflammation are increased when there is a deficiency of inhibitors of T cell activation, including the PD-1/PD-L1 and PD-L2 pathways and regulatory T cells [Adaptive immunity in atherogenesis: new insights and therapeutic approaches. Lichtman A H, Binder C J, Tsimikas S, Witztum J L. s.l.: J Clin Invest, 2013, Vol. 123, pp. 27-36].
Baixeras et al, supra, characterized the cellular distribution of LAG3 in a number of cell lines and demonstrated that LAG3 resided within lipid rafts. Subsequently, Woo et al. reported the intracellular distribution of LAG3 and found that LAG3 was equally distributed between intracellular compartments and the plasma membrane. [Woo S-R, Li N, Bruno T C, Forbes K, Brown S, Workman C, Drake C G, Vignali D A A. Differential subcellular localization of the regulatory T-cell protein LAG-3 and the coreceptor CD4. Eur J Immunol 2010; 40:1768-1777]
By using flow cytometry, the present inventor confirmed that low levels of LAG3 were detected on the cell surface of rs10846744 risk C expressing cells regardless of stimulation conditions. However, LAG3 was expressed on the cell surface in unstimulated rs10846744 reference G cells and its levels increased significantly after stimulation. These results in EBV transformed B cells are in contrast to those reported by Woo et al, supra, in that they reported that LAG3 was expressed on the surface only in activated T cells.
It is also known that lipid raft signaling is essential for B cell activation. [Simons K, Toomre D. Lipid rafts and signal transduction. [Nat Rev Mol Cell Biol 2000; 1:31-39] Specifically, stimulation of the B cell receptor (BCR) initiates phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic tails of CD79A and CD79B (transmembrane immunoglobulin (Ig) receptor associated with Ig-alpha/Ig-beta heterodimers) [Schamel W W, Reth M. Monomeric and oligomeric complexes of the B cell antigen receptor. Immunity. 2000; 13:5-14] Phosphorylation of ITAMs serve as docking sites for Syk, which is mediated by different Src family kinases (SFKs) including Fyn, Blk, and Lyn [Takata M, Sabe H, Hata A, Inazu T, Homma Y, Nukada T, Yamamura H, Kurosaki T. Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways. EMBO J. 1994; 13:1341-9.]. Lyn is the major protein involved in lipid raft signaling upon B cell activation [Simons, supra]. This activation initiates the coordinate assembly of the “signalosome”, composed of a variety of intracellular signaling molecules and includes Btk, phosphatidylinositol 3-kinase (PI3K) and PLCγ2 [Blix E S, Irish J M, Husebekk A, Delabie J, Forfang L, Tierens A M, Myklebust J H, Kolstad A. Phospho-specific flow cytometry identifies aberrant signaling in indolent B-cell lymphoma. BMC Cancer 2012:12:478.] PLCγ2 is the predominant isoform expressed in human B lymphocytes [Coggeshall K M, McHugh J C, Altman A. Predominant expression and activation-induced tyrosine phosphorylation of phospholipase C-gamma 2 in B lymphocytes. Proc Natl Acad Sci USA. 1992; 89:5660-4.] It is also indispensable for BCR-mediated phosphoinositol hydrolysis and the subsequent biochemical events including PKC activation [Sugawara H, Kurosaki M, Takata M, Kurosaki T. Genetic evidence for involvement of type 1, type 2 and type 3 inositol 1, 4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor. EMBO J. 1997; 16:3078-88].
MHC class II is the main ligand to LAG3, and the latter binds to the former with high affinity where it negatively regulates cellular proliferation, activation, and homeostasis of T cells, and has been reported to play a role in Treg suppressive function. By contrast, signaling through MHC class II in lipid raft microdomains on a subset of dendritic cells after it is bound by soluble LAG3 (sLAG3) results in dendritic cell activation. The inventor has now discovered that cellular LAG3 in lymphocytes is causal in regulating the phosphosignaling cascade. This is a distinct and novel function of LAG3, independent of its previous known function of binding to MHC class II receptors.
However, the major apolipoprotein associated with HDL particles, apoA-1, has been shown to inhibit inflammatory cytokine production by inhibiting activation of monocytes by T lymphocytes [Hyka N, Dayer J-M, Modoux C, Kohno T, Edwards III C K, Roux-Lombard P, Burger D. Apolipoprotein A-I inhibits the production of interleukin-1β and tumor necrosis factor-α by blocking contact-mediated activation of monocytes by T lymphocytes. Blood 2001; 97:2381-2389]. Specifically, Hyka et al. observed that apoA-I inhibited cytokine production from stimulated monocytes by first binding to a surface factor, which suggests the possibility that apoA-I might interact with surface LAG3.
The significant association of SCARB1 variant, rs10846744, with coronary heart disease (CHD) was shown in Manichaikul et al (Arterioscler Thromb Vase Biol 2012; 32:1991-1999). However, previous analyses do not show that rs10846744 is directly associated with SCA and incident CVD. This is because, as the present inventor has found, LAG3 is an important immune regulator that mediates the association of rs10846744 with atherosclerotic disease and CVD. LAG3 protein expression on effector and regulatory T cells may inhibit T cell receptor (TCR)-mediated activation by blocking TCR interaction with MHC class II, and LAG3 protein on plasmacytoid DCs may indirectly suppress effector T cells by other mechanisms. LAG3 protein deficiency may lead to enhanced inflammasome mediated IL-1β and IL-I 18 production by DCs and macrophages, two cytokines that skew T cell differentiation to inflammatory phenotypes that possess specialized cytokine potential [The interleukin-1 family: back to the future. Garlanda C, Dinarello C A, Mantovani A. s.l.: Immunity, 2013, Vol. 39, pp. 1003-1018].
Therefore, LAG3 protein deficiency in mice (Lag3) and humans (LAG3) is predicted to enhance pro-atherogenic T cell responses to hypercholesterolemia and lead to increased plaque inflammation and/or increased plaque development.
It has not been obvious that deficiency of LAG-3 protein due to genetic variations exists in humans and that it is significantly associated with atherosclerosis, other chronic inflammatory diseases, cardiovascular and/or noncardiovascular mortality. Based on the mediator role that LAG3 plays in CVD and other chronic inflammatory diseases, the present invention discloses a method for using LAG3 expression profiling as a biomarker for assessing patients at risk of CVD based on certain SCARB1 and LAG-3 gene variations and other genetic and non-genetic factors that increase risk for clinically significant atherosclerosis, other chronic inflammatory diseases, chronic inflammatory diseases, dysfunctional HDL, cardiovascular and/or non-cardiovascular morbidity and mortality and for ameliorating said risk with a novel recombinant LAG3 companion therapeutic.
The therapeutic activity of IMP321 (the human dimeric soluble form of LAG3) is well-known in pre-clinical as well as clinical studies. It has been shown that the combination of hLAG3—Ig as an adjuvant with chemotherapeutic agents is superior to either treatment on its own.
Furthermore, a LAG3 blockade may be combined with blockade of other inhibitory receptors, such as PD-1, resulting in enhanced T cell activity and protection from disease. In addition, the therapeutic activity of soluble recombinant dimeric LAG3 protein is also known in several respects. [Sierro et al., “The CD4-like molecule LAG-3, biology and therapeutic applications”, Section 3]. In humans, recombinant LAG3 induces DC activation and provides immune adjuvant activity (in contrast to the inhibitory activity of the membrane-bound form of LAG-3). Andreae et al., “Maturation and activation of dendritic cells induced by lymphocyte activation gene-3 (CD223)”, J Immunol, 168:3874-80 (2002).
U.S. Pat. No. 6,410,509 to Triebel (Institut Gustave-Roussy) issued Jun. 25, 2002 shows the use of hLAG3 as adjuvant for vaccination and in cancer treatment, the systemic administration of soluble hLAG3 directly inducing an inhibition of in vivo tumor growth (see Example IV). Similarly, United States Patent Application 20110008331 to Triebel (Immutep) published Jan. 13, 2011 shows the periodic use of a recombinant LAG3 to boost a monocyte-mediated immune response, in particular to elicit an increase in the number of monocytes in blood. The application notes the discovery “entirely unexpectedly” that human LAG3 or derivatives thereof when inoculated into patients with highly malignant tumors induced a potent immunity which is monocyte dependent. The induced immunity manifests itself by a significant increase in blood monocyte counts. Despite the use of hLAG3 as a vaccine adjuvant to boost T-cell counts in known cancer patients, no one has yet been motivated to peremptorily screen for a LAG3 deficiency marker and, if found, ameliorate the consequent risk by treating prospective patients with recombinant human LAG3 by periodic administration of at different time points. The present inventor does this by pre-screening patients at risk for cardiovascular disease, other chronic inflammatory diseases, cardiovascular and/or non-cardiovascular morbidity and mortality using specific 2-point expression profiles (combinations of mutations and/or non-genetic causes, e.g., SCARB1 rs10846744 mutation and/or LAG-3 rs870849 or other genetic or non-genetic causes and a mutation), followed by a tailored therapeutic regimen using recombinant human lymphocyte activation gene-3 as a companion therapeutic.